Feature: Visualising the cellular membrane

The basic components for synthesising, assembling and operating a higher life form come wrapped in a diaphanous, greasy film just nanometers thick. Only a decade ago many biologists regarded the eukaryotic cell’s external or plasma membrane as little more than a passive stage for the molecular theatre of life. Diagrams in biology texts represented the plasma membrane as simple bilayer of phospholipid molecules, oriented with hydrophobic heads pointing outwards and joined by their tails.

Associate Professor Katharina Gaus, head of the Cellular Membrane Biology Laboratory at the University of New South Wales Centre for Vascular Research, says the reality is very different: the plasma membrane is not homogeneous but a shifting mosaic of hundreds of different lipids that is actively involved in cell function. She believes the cell’s lipid ‘coat of many colours’ fine-tunes the functions of the multitude of protein molecules embedded in the plasma membrane: signalling receptors, ion channels, cell adhesion molecules and anchorage points for the internal cytoskeleton.

In 2003, Gaus was the lead author of a seminal paper in Proceedings of the National Academy of Science that reported the first direct visualisation of lipid rafts in cell membranes via high-resolution fluorescence microscopy. The lipid rafts consist of highly condensed patches of specific lipids such as cholesterol and sphingolipids. In white blood cells, semi-rigid rafts of cholesterol cover about 10 to 15 per cent of the cell surface. Membrane protrusions called filopodia, adhesion points and cell-to-cell contacts are highly enriched in the lipid rafts, suggesting that cell-membrane proteins are anchored within the lipid rafts, which influence their function.

Gaus will describe her group’s latest findings at the Australian Peptide Association’s annual conference, Peptide Australia 2008, held on Stradbroke Island in October.

“I’m very much a fundamental scientist,” says Gaus. “I’m trying to understand the cell membrane at the level of processes that occur within it rather than in terms of the structure and function of individual molecules. Before 2003, much of the debate around lipid rafts was around whether they existed and, if so, how they could be characterised, and what technology was needed to do that.

“The debate became almost academic, and revolved around semantics: if such an entity existed, could it be called a lipid raft or something else. It wasn’t really advancing the field. So we tried to step outside the debate and ask whether membrane organisation influenced protein function, as had been postulated. We set out to link particular membrane domains to specific cell functions, such as T cell activation in an immune response.

“We started by setting up mass spectrometry to characterise every lipid, then devised experiments to determine whether manipulating cholesterol levels in the cell changed the abundance of other lipids. Did changing one lipid change the abundance of other lipids? And how does that change membrane domains and, as a consequence, T cell signalling?

“It was a bean-counting exercise, but we needed information on the abundance of various lipids and how they are distributed in the cell membrane, to understand how they might influence protein distribution and function.”

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An abundance of lipids

Gaus says approximately a third of the 24,000-odd human genes code for membrane-associated proteins and mammalian cells have the capacity to synthesise around 1,000 different lipids.

No cell produces the entire complement of lipids, but the T cells that Gaus and her colleagues employ as models in their research typically have plasma membranes consisting of up 400 different lipids, not counting low-abundance lipids that may escape detection. It’s an intriguing question as to why cells need so many membrane lipids to function, but Gaus says that with so many proteins and lipids there is potential for myriad interactions between them.

“To understand them, you need a way of describing how proteins are located within the membrane,” she says. “For that we use fluorescence microscopy – we attach fluorophores to proteins of interest or we stain the whole membrane to get an idea of how the proteins and lipids are distributed relative to each other.”

The field is still in its infancy; how the lipid rafts influence the function of their protein passengers is “a million-dollar question,” says Gaus. “But we are convinced it’s a two-way interaction. When you trigger a receptor protein in the membrane and it starts to signal inside the cell, we believe it influences the lipid around it and the lipid affects its behaviour in turn.”

The fact that cells regulate their lipid levels so tightly points to their integral role in protein function. It seems the old advertising slogan ‘oils ain’t oils’ applies to life, as well as lubrication. Cholesterol is crucial to normal membrane permeability and function but, because it is not available from plants, mammals synthesise their own. Gaus says even when cells are exposed to high serum cholesterol levels from a high-fat diet, cells need to maintain a homeostasis so that they can function properly.

But humans are susceptible to a variety of inherited lipid metabolism disorders with serious or even lethal health consequences. “Obese patients often have a dysregulated immune system, which involves a hyperactive or under-active T cells. Hyperactive T cells can cause auto-immune disorders, while underactive T cells leave them vulnerable to infections.”

Gaus’s studies of protein-lipid interactions have focused on the early events after a pathogen-derived peptide binds to a receptor, triggering a signalling cascade inside the cell and activating that cell so it can participate in an immune response. They are investigating how kinases are recruited to the receptor’s intracellular domain and the downstream events that follow. “Does reorganising lipids in the membrane around the receptor have ripple-down effects on the signalling cascade?”

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Super resolution

Gaus says while biochemistry is useful in identifying interactions between molecules at the broad level, analysing cell extracts does not reveal the dynamics of such interactions in living cells. “Fluorescence microscopy is our method of choice because it allows us to image living cells and determine how reorganising membrane lipids affects signalling,” she says.

But there has been one huge problem with this microscopy approach to date. “The diffraction limit prevents ordinary light microscopes resolving detail smaller than about 200-300 nanometres. If two protein molecules are close together, we can’t distinguish between them. Protein molecules are around 10-20 nanometres wide, so if we want to image them, we’re well below the resolution limit for light microscopy. At the moment, we’re setting up a new range of microscopy tools, called super-resolution fluorescence microscopy, which have a resolution of below 20 nanometers. This allows us to image cell membranes truly on a molecular scale.

“It’s a fantastic time to be in fluorescence microscopy – the technology has made huge leaps. Not only can we observe the dynamic behaviour of a signalling protein, we can resolve its location with high accuracy and even count the number of molecules within that region of the cell membrane.”

Although her research falls at the fundamental end of the spectrum, it has yielded a discovery with exciting clinical implications. Gaus and her colleagues have been using T cells to study how membrane lipid composition influences signal transduction by T cell receptors. They believe they have identified a fundamental signalling mechanism that is disrupted in obese subjects and are excited that the insight could lead to a therapy to restore a normal immune response in obese patients.